Methods and materials for treating cancer

ABSTRACT

This document provides methods and materials for treating cancers including renal cancer (e.g., renal cell carcinoma) as well as ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung, and thyroid cancers and melanoma. For example, methods and material for using one or more inhibitors of an SCD1 polypeptide to treat renal cell carcinoma (e.g., clear cell renal cell carcinoma (ccRCC)) or to increase the efficacy of a renal cell carcinoma treatment are provided. In addition, this document provides methods and materials for using elevated SCD1 expression levels in diseased tissues as an indication that an SCD1 inhibitor can be used as an appropriate therapeutic to ameliorate the disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. 371 ofInternational Application No. PCT/US2013/029688, having an internationalfiling date of Mar. 7, 2013, which claims priority to U.S. ProvisionalApplication Ser. No. 61/607,961, filed on Mar. 7, 2012, each of which isincorporated by reference in its entirety herein.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This invention was made with government support under CA104505 andCA136665 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in treatingcancer, for example, renal cell carcinoma, ovarian, breast, prostate,colon, pancreatic, bladder, liver, lung, thyroid cancers, and melanoma.For example, this document provides methods and material for using oneor more inhibitors of a stearoyl-Coenzyme A desaturase 1 (SCD1)polypeptide to treat cancer.

2. Background Information

The incidence and deaths caused by renal cell carcinoma are increasingin the United States. Indeed, mortality from renal cell carcinoma hasincreased over 37% since 1950.

SUMMARY

This document provides methods and materials for treating cancer, forexample, renal cell carcinoma, ovarian, breast, prostate, colon,pancreatic, bladder, liver, lung, thyroid cancers, and melanoma. Forexample, this document provides methods and material for using one ormore inhibitors of an SCD1 polypeptide to treat renal cell carcinoma(e.g., clear cell renal cell carcinoma (ccRCC)) or to increase theefficacy of a renal cell carcinoma treatment. As described herein, SCD1polypeptides are overexpressed in certain cancer cells and are involvedin the survival or proliferation of cancer cells. For example, reducingexpression of renal cell carcinoma cells can result in reducedproliferation of renal cell carcinoma cells with minimal or no reductionin proliferation of normal kidney cells. In some cases, one or moreinhibitors of an SCD1 polypeptide can be used to reduce the number ofcancer cells within a mammal (e.g., a human). In some cases, one or moreinhibitors of an SCD1 polypeptide can be used to increase the efficacyof a cancer treatment. For example, one or more inhibitors of an SCD1polypeptide can be used to increase the efficacy of a renal cellcarcinoma treatment (e.g., treatment with Nexavar®, Sutent®, Torisel®,Afinitor®, and interleukin-2).

In general, one aspect of this document features a method for reducingthe number of renal cell carcinoma cells within a mammal. The methodcomprises, or consists essentially of, administering, to the mammal, aninhibitor of an SCD1 polypeptide under conditions wherein the number ofviable renal cell carcinoma cells present within the mammal is reduced.The mammal can be a human. The administration can be an intratumoral,oral, intraperitoneal, intramuscular, or intravenous administration. Theinhibitor can be A939572, MK-8245, CVT-11127, MF-152, or HYR-061. Inanother embodiment, one or more inhibitors of an SCD1 polypeptide can beadministered with one or more inhibitors of a mTor polypeptide.Non-limiting examples of such inhibitors include sirolimus (RAPAMUNE®),temsirolimus (CCI-779), everolimus (RAD001), and ridaforolimus(AP-23573).

In another aspect, this document features a method for reducing thenumber of renal cell carcinoma cells within a mammal. The methodcomprises, or consists essentially of, administering, to the mammal, acomposition under conditions wherein the number of viable renal cellcarcinoma cells present within the mammal is reduced, wherein thecomposition comprises the ability to reduce SCD1 mRNA expression or SCD1polypeptide expression. The mammal can be a human. The administrationcan be an intratumoral, oral, intraperitoneal, intramuscular, orintravenous administration. The composition can comprise a nucleic acidconstruct having the ability to express an shRNA directed against SCD1nucleic acid.

In another aspect, this document features a method for reducing thenumber of cancer cells overexpressing an SCD1 polypeptide within amammal. The method comprises, or consists essentially of, administering,to the mammal, an inhibitor of an SCD1 polypeptide under conditionswherein the number of viable cancer cells overexpressing an SCD1polypeptide present within the mammal is reduced. Non-limiting examplesof cancers include renal cell carcinoma, ovarian, breast, prostate,colon, pancreatic, bladder, liver, lung, thyroid cancers, and melanoma.

In another aspect, this document features a method for reducing thenumber of cancer cells overexpressing an SCD1 polypeptide within amammal. The method comprises, or consists essentially of, administering,to the mammal, a composition under conditions wherein the number ofviable cancer cells overexpressing an SCD1 polypeptide present withinthe mammal is reduced, wherein the composition comprises the ability toreduce SCD1 mRNA expression or SCD1 polypeptide expression. Non-limitingexamples of cancers include renal cell carcinoma, ovarian, breast,prostate, colon, pancreatic, bladder, liver, lung, thyroid cancers, andmelanoma.

In another aspect, this document features a method for identifying amammal having cancer cells responsive to treatment with an inhibitor ofan SCD1 polypeptide. The method comprises, or consists essentially of,(a) detecting the presence of cancer cells expressing an elevated levelof an SCD1 mRNA or an SCD1 polypeptide, and (b) classifying the mammalhas having cancer cells responsive to treatment with the inhibitor of anSCD1 polypeptide. The method can comprise measuring SCD1 mRNA expressionusing real time PCR. The method can comprise measuring SCD1 polypeptideexpression using an immunohistochemical technique. The method cancomprise measuring SCD1 polypeptide expression using a Western blotanalysis. Non-limiting examples of cancers include renal cell carcinoma,ovarian, breast, prostate, colon, pancreatic, bladder, liver, lung,thyroid cancers, and melanoma.

In a further aspect, this document features a method for reducing thenumber of cancer cells within a mammal. The method comprises, orconsists essentially of, administering, to said mammal, an inhibitor ofan SCD1 polypeptide and an inhibitor of an mTor polypeptide underconditions wherein the number of viable cancer cells present within saidmammal is reduced. In some cases, the inhibitor of an mTor polypeptidecan be sirolimus (RAPAMUNE®), temsirolimus (CCI-779), everolimus(RAD001), or ridaforolimus (AP-23573). In certain cases, the mammal is ahuman. In some cases, the administration is an intratumoral, oral,intraperitoneal, intramuscular, or intravenous administration. In somecases, the inhibitor of an SCD1 polypeptide is A939572, MK-8245,CVT-11127, MF-152, or HYR-061. Non-limiting examples of cancer cellsinclude one or more of ovarian cancer, breast cancer, prostate cancer,colon cancer, renal cancer, pancreatic cancer, bladder cancer, livercancer, lung cancer, thyroid cancer, and melanoma.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph plotting SCD1 mRNA levels in ccRCC tissue and matchednormal tissue across stages I-IV. FIG. 1B contains photographs ofrepresentative ccRCC tissue and matched normal tissue stained for SCD1polypeptide expression. FIG. 1C is a graph plotting SCD1 mRNA levels innormal renal epithelial cell lines (347N, 355N, 359N, 360N, 365N, and366N) versus ccRCC cell lines. FIG. 1D contains photographs of a Westernblot analysis of SCD1 polypeptide expression by normal cell lines andccRCC cell lines.

FIGS. 2A and 2D shows the knockdown of SCD1 in ccRCC as shown bydecrease in both (A) mRNA and (D) protein expression using two separatelentiviral constructs shSCD780 and shSCD1200. FIGS. 2B and 2C showproliferation in (B) A498 and Caki1 ccRCC cell lines and (C) NRE samplesof NT versus shSCD lentiviral infected cells. FIG. 2D containsphotographs of an immunoblot for Poly-ADP ribose polymerase (PARP)cleavage and SCD1 expression in A498 and Caki1 cell lines.

FIG. 3. Anti-proliferative and apoptotic induction via loss of SCD1expression can be rescued with addition of oleic acid (OA-BSA). FIG. 3Ais a bar graph showing proliferation for SCD1 and PARP cleavage in Caki1and A498 NT versus shSCD with or without OA-BSA supplementation. FIG. 3Bcontains photographs of a Western blot analysis for SCD1 and PARPcleavage in Caki1 and A498 NT versus shSCD with or without OA-BSAsupplementation. FIG. 3C contains photographs of a phase-contrastmicroscopy representative ccRCC cell (Caki1) confluence at day 5 ofproliferation assay with different treatment conditions.

FIG. 4. Treatment of ccRCC cells with a small molecule SCD1 inhibitor,A939572, inhibits cell growth and induces apoptosis. FIG. 4A is a linegraph showing cell proliferative response to dose out of A939572 inCaki1, A498, Caki2, and ACHN ccRCC cell lines. FIG. 4B is a bar graphdisplaying ccRCC proliferation rescue with OABSA in A939572 treatedccRCC cell lines. FIG. 4C contains photographs of a Western blotanalysis for PARP cleavage in A939572 treated vs. control, as well asOA-BSA rescue in ccRCC cell lines. FIG. 4D contains representative phasecontrast images of A939572 treated ccRCC cells (A498)+/−OA-BSA rescue atday 5.

FIG. 5. Inhibition of SCD1 activity in ccRCC induces cell death mediatedby endoplasmic reticulum stress response. FIG. 5A contains photographsof a Western blot analysis for expression of ER stress markers: BiP,CHOP, and spliced XBP1 in response to A939572 treatment or lentiviralsilencing of SCD1 in Caki1 and A498. FIG. 5B provides bar graphs showingQPCR analysis of ER stress gene expression in Caki1 and A498 cellstreated with A939572 or shSCD lentivirus+/−OA-BSA rescue. FIG. 5Cprovides bar graphs showing relative luciferase activity of ER stresspxATF6-GL3 (UPR) luciferase reporter transfected in Caki1 and A498 cellstreated with A939572 or shSCD lentivirus+/−OA-BSA supplementation.

FIG. 6. Treatment of ccRCC cells with SCD1 inhibitor in combination withthe mTOR inhibitor Temsirolimus synergistically inhibits tumor cellgrowth in vivo. FIG. 6A is a line graph illustrating in vivo tumorgrowth analysis and animal weight of A498 ccRCC subcutaneous xenograftsin female athymic nude mice treated with A939572 and Temsirolimus aloneor in combination versus placebo control (n=10 per group). FIG. 6Bcontains photographs of IHC of tissue harvested from treatment groupsstained for Ki67 and CC3 (quantitated by N-score), CD31 (quantitated byI-score), and phospho-mTOR (quantitated by H-score). Average groupscores+/−the standard error are reported for each stain. FIG. 6Ccontains photographs of Western blot and quantitation of CHOP expressionin all four treatment groups. FIG. 6D is an illustration of proposedSCD1 activity in ccRCC model: inhibition of SCD1 blocks desaturation ofSFA resulting in an accumulation of SFA species which trigger the ERstress response.

FIGS. 7A-D are line graphs comparing cell number to dose of A939572 orGemicitabine in MiaPaca and pancreatic cancer cells. FIG. 7E containsphotographs of Western Blot and quantitation of SCD1 and beta-actinexpression.

FIGS. 8A-B are line graphs comparing cell number to dose of A939572 orSorafenib in SNU449 liver cancer cells. FIG. 8C contains photographs ofWestern Blot and quantitation of SCD1 and beta-actin expression.

FIGS. 9A-C are line graphs comparing cell number to dose of A939572 orTemodar in A375 AND Mela 11 melanoma cancer cells. FIG. 9D containsphotographs of Western Blot and quantitation of SCD1 and beta-actinexpression.

FIGS. 10A-B are line graphs comparing cell number to dose of A939572 orCapecitabine in CaCo2 and HT29 colon cancer cells. FIG. 10C containsphotographs of Western Blot and quantitation of SCD1 and beta-actinexpression.

FIGS. 11A-B are line graphs comparing cell number to dose of A939572 orcisplatin in T24 and HT1276 bladder cancer cells. FIG. 11C containsphotographs of Western Blot and quantitation of SCD1 and beta-actinexpression.

FIGS. 12A-B are line graphs comparing cell number to dose of A939572 orcisplatin in BCJ4T bladder cancer cells. FIG. 12C contains photographsof Western Blot and quantitation of SCD1 and beta-actin expression.

FIGS. 13A-B are line graphs comparing cell number to dose of A939572 orTaxol in KTC3 and FF1 anaplastic thyroid cancer cells. FIG. 13C containsphotographs of Western Blot and quantitation of SCD1 and beta-actinexpression.

FIGS. 14A-B are line graphs comparing cell number to dose of A939572 orTaxol in A549 and CaLu-1 lung cancer cells. FIG. 14C containsphotographs of Western Blot and quantitation of SCD1 and beta-actinexpression.

FIGS. 15A-B are line graphs comparing cell number to dose of A939572 orTaxol in OVCA420 and HOVTax2res ovarian cancer cells. FIG. 15C containsphotographs of Western Blot and quantitation of SCD1 and beta-actinexpression.

FIG. 16A is a line graph comparing cell number to dose of A939572 inMCF-7 (ER+/PR+), MDA-231 (triple negative) and T47D (PR+) breast cancercells. FIG. 16B contains photographs of Western Blot and quantitation ofSCD1 and beta-actin expression.

FIG. 17A is a line graph comparing cell number to dose of A939572 inDU-145 prostate cancer cells. FIG. 17B contains photographs of WesternBlot and quantitation of SCD1 and beta-actin expression.

FIG. 18A is a bar graph illustrating SCD1 protein expression in variouscancer cell lines. FIG. 18B contains photographs of Western Blot andquantitation of SCD1 and beta-actin expression.

FIG. 19A is a bar graph illustrating SCD1 protein expression in variouscancer cell lines. FIG. 19B contains photographs of Western Blot andquantitation of SCD1 and beta-actin expression.

FIGS. 20-22 provide structures for exemplary SCD1 inhibitors.

DETAILED DESCRIPTION

This document provides methods and materials for treating cancer, forexample, for example, renal cell carcinoma, ovarian, breast, prostate,colon, pancreatic, bladder, liver, lung, thyroid cancers, and melanoma.In some embodiments, this document provides methods and material forusing one or more inhibitors of an SCD1 polypeptide to treat cancer(e.g., clear cell renal cell carcinoma (ccRCC)) or to increase theefficacy of a cancer treatment.

As described herein, one or more (e.g., one, two, three, four, or more)inhibitors of an SCD1 polypeptide can be administered to a mammal (e.g.,a human) having cancer (e.g., renal cancer) under conditions wherein thenumber of cancer cells within the mammal is reduced. In someembodiments, one or more (e.g., one, two, three, four, or more)inhibitors of an SCD1 polypeptide can be administered to a mammal (e.g.,a human) having renal cancer (e.g., ccRCC) under conditions wherein thenumber of renal cancer cells within the mammal is reduced.

An SCD1 polypeptide can be a human SCD1 polypeptide having the aminoacid sequence set forth in GenBank® Accession No. 000767 (GI No.21431730) or a human SCD1 polypeptide encoded by nucleic acid having thenucleic acid sequence set forth in GenBank® Accession No. AF097514.1 (GINo. 4808600). Examples of inhibitors of an SCD1 polypeptide include,without limitation, inhibitory anti-SCD1 polypeptide antibodies, siRNAmolecules, shRNA molecules, nucleic acid vectors designed to expresssiRNA or shRNA molecules, anti-sense molecules, and small moleculeantagonists such as A939572 (Biofine International Inc., Urvashi et al.,Mol. Cancer Res., 9:1551 (2011); Bristol-Myers Squibb R&D, Roongta etal., Mol. Cancer Res., 9(11):1551-61 (2011)), MK-8245 (Merck ResearchLaboratories, Oballa et al., J. Med. Chem., 54(14):5082-96 (2011)),CVT-11127, MF-152 (Merck, Li et al., Bioorganic & Medicinal ChemistryLetters, 19:5214 (2009)), LCF369, CVT-11,563, CVT-12,012, DSR-4029, andGSK993 (Uto et al., Eur. J. Med. Chem., 45:4788-4796 (2010)), MF-438(Leger, S. et al., Bioorg Med Chem Lett. 20(2):499-502 (2010)), andHYR-061 (Medchem Express, Koltun et al., Bioorganic & MedicinalChemistry Letters, 19(7):2048-2052 (2009), and Xin et al., Bioorganic &Medicinal Chemistry Letters, 18(15):4298-4302 (2008)). In some cases, aninhibitor of an SCD1 polypeptide can be an inhibitor described elsewhere(Igal, Carcinogenesis, 31(9):1509-1515 (2010); Oballa, J. Med. Chem.,54:5082-5096 (2011); Li et al., Bioorganic & Medicinal ChemistryLetters, 19:5214-5217 (2009); Uto et al., Eur. J. Med. Chem.,46:1892-1896 (2011); Uto et al., Eur. J. Med. Chem., 45:4788-4796(2010); Liu, G. Expert Opin Ter Pat, 19(9):1169-91 (2009); Powell, D.A., Bioorg Med Chem Lett. 20(22):6366-9 (2010), Mason P, et al., PLoSONE 7(3): 33823 (2012), and Roongta et al., Mol. Cancer Res.,9:1551-1561 (2011)).

Cancers that may be treated by an inhibitor of an SCD1 polypeptide,compositions and methods described herein include, but are not limitedto, the following:

-   -   Breast cancers, including, for example ER⁺ breast cancer, ER⁻        breast cancer, her2⁻ breast cancer, her2⁺ breast cancer, stromal        tumors such as fibroadenomas, phyllodes tumors, and sarcomas,        and epithelial tumors such as large duct papillomas; carcinomas        of the breast including in situ (noninvasive) carcinoma that        includes ductal carcinoma in situ (including Paget's disease)        and lobular carcinoma in situ, and invasive (infiltrating)        carcinoma including, but not limited to, invasive ductal        carcinoma, invasive lobular carcinoma, medullary carcinoma,        colloid (mucinous) carcinoma, tubular carcinoma, and invasive        papillary carcinoma; and miscellaneous malignant neoplasms.        Further examples of breast cancers can include luminal A,        luminal B, basal A, basal B, and triple negative breast cancer,        which is estrogen receptor negative (ER⁻), progesterone receptor        negative, and her2 negative (her2⁻). In some embodiments, the        breast cancer may have a high risk Oncotype score;    -   lung cancers, including, for example, bronchogenic carcinoma,        e.g., squamous cell, undifferentiated small cell,        undifferentiated large cell, and adenocarcinoma; alveolar and        bronchiolar carcinoma; bronchial adenoma; sarcoma; lymphoma;        chondromatous hamartoma; and mesothelioma;    -   genitourinary tract cancers, including, for example, cancers of        the kidney, e.g., adenocarcinoma, Wilm's tumor (nephroblastoma),        lymphoma, and leukemia; cancers of the bladder and urethra,        e.g., squamous cell carcinoma, transitional cell carcinoma, and        adenocarcinoma; cancers of the prostate, e.g., adenocarcinoma,        and sarcoma; cancer of the testis, e.g., seminoma, teratoma,        embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma,        interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid        tumors, and lipoma;    -   liver cancers, including, for example, hepatoma, e.g.,        hepatocellular carcinoma; cholangiocarcinoma; hepatoblastoma;        angiosarcoma; hepatocellular adenoma; and hemangioma;    -   gynecological cancers, including, for example, cancers of the        uterus, e.g., endometrial carcinoma; cancers of the cervix,        e.g., cervical carcinoma, and pre tumor cervical dysplasia;        cancers of the ovaries, e.g., ovarian carcinoma, including        serous cystadenocarcinoma, epithelial cancer, mucinous        cystadenocarcinoma, unclassified carcinoma, granulosa thecal        cell tumors, Sertoli Leydig cell tumors, dysgerminoma, and        malignant teratoma; cancers of the vulva, e.g., squamous cell        carcinoma, intraepithelial carcinoma, adenocarcinoma,        fibrosarcoma, and melanoma; cancers of the vagina, e.g., clear        cell carcinoma, squamous cell carcinoma, botryoid sarcoma, and        embryonal rhabdomyosarcoma; and cancers of the fallopian tubes,        e.g., carcinoma;    -   skin cancers, including, for example, malignant melanoma, basal        cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles        dysplastic nevi, lipoma, angioma, dermatofibroma, keloids,        psoriasis; and    -   adrenal gland cancers, including, for example, neuroblastoma.

In some cases, one or more (e.g., one, two, three, four, or more)inhibitors of an SCD1 polypeptide can be used as described herein totreat cancer, including renal cancer, ovarian, breast, prostate, colon,pancreatic, bladder, liver, lung, and thyroid cancers as well asmelanoma.

For example, a human having cancer can be administered one or moreinhibitors of an SCD1 polypeptide under conditions that result inreduced tumor size or stable disease. In some cases, one or more (e.g.,one, two, three, four, or more) inhibitors of an SCD1 polypeptide can beused as described herein to increase the efficacy of a cancer treatment.In some embodiments (e.g., when compositions comprising one or more(e.g., one, two, three, four, or more) inhibitors of an SCD1 polypeptideare administered in conjunction with another anticancer agent), one cancreate a synergistic effect among the agents administered and therebyimprove the outcome for a patient. In some embodiments, one or more(e.g., one, two, three, four, or more) inhibitors of an SCD1 polypeptide(or a pharmaceutically acceptable salt form thereof) can be administeredin combination with (i.e., before, during, or after) administration of apain relief agent (e.g., a nonsteroidal anti-inflammatory drug such ascelecoxib or rofecoxib), an antinausea agent, or an additionalanticancer agent (e.g., paclitaxel, docetaxel, doxorubicin,daunorubicin, epirubicin, fluorouracil, melphalan, cis-platin,carboplatin, cyclophosphamide, mitomycin, methotrexate, mitoxantrone,vinblastine, vincristine, ifosfamide, teniposide, etoposide, bleomycin,leucovorin, taxol, herceptin, avastin, cytarabine, dactinomycin,interferon alpha, streptozocin, prednisolone, irinotecan, sulindac,5-fluorouracil, capecitabine, oxaliplatin/5 FU, abiraterone, letrozole,5aza/romidepsin, or procarbazine). In certain embodiments, theanticancer agent is paclitaxel or docetaxel. In other embodiments, theanticancer agent is cisplatin or irinotecan.

For example, a human having ccRCC can be administered one or moreinhibitors of an SCD1 polypeptide under conditions that result inreduced tumor size or stable disease. In some cases, one or more (e.g.,one, two, three, four, or more) inhibitors of an SCD1 polypeptide can beused as described herein to increase the efficacy of a renal cellcarcinoma treatment. Examples of such renal cell carcinoma treatmentsinclude, without limitation, treatment with Nexavar®, Sutent®, Torisel®,Afinitor®, or interleukin-2.

In some cases, one or more (e.g., one, two, three, four, or more)inhibitors of an SCD1 polypeptide can be used as described herein can beused in combination with one or more (e.g., one, two, three, four, ormore) inhibitors of mammalian target of rapamycin (mTor) polypeptide.Non-limiting examples of mTor inhibitors include: sirolimus (RAPAMUNE®),temsirolimus (CCI-779), everolimus (RAD001), ridaforolimus (AP-23573).

Accordingly, provided herein is a method for reducing the number ofcancer cells within a mammal, wherein the method comprisesadministering, to the mammal, an inhibitor of an SCD1 polypeptide and aninhibitor of an mTor polypeptide under conditions wherein the number ofviable cancer cells present within said mammal is reduced. In someembodiments, the one or more mTor inhibitor can include a standard ofcare drug for a particular cancer cell type. For example, an SCD1inhibitor can be administered with pacliltaxel and/or platin (cisplatin,carboplatin, or oxaliplatin) for the treatment of ovarian cancer. Insome embodiments, the following standard of care drugs can be combinedwith an SCD1 inhibitor for the following cancers:

Lung—paclitaxel

Colon—capecitabine

Breast

-   -   Metastatic breast—capecitabine, paclitaxel, and/or gemcitabine    -   Hormonally responsive breast—aromatase inhibitors such as        letrazole and/or antiestrogens such as tamoxifen    -   HER2 positive—Herceptin

Melanoma—temodar, and/or BRAF inhibitors

Prostate—abiraterone

Bladder—gemcitabine and/or paclitaxel

Thyroid—paclitaxel and/or cisplatin

Pancreatic—gemcitabine

Liver—sorafanib

In some embodiments, the combination of one or more inhibitors of anSCD1 polypeptide and one or more inhibitors of mTor exhibit asynergistic response. In some embodiments, the one or more inhibitors ofan SCD1 polypeptide can be administered before, during, or afteradministration of the one or more inhibitors of mTor.

An inhibitor of an SCD1 polypeptide can also be administered to asubject in combination with surgical methods to treat cancers, e.g.,resection of tumors. The inhibitor can be administered to the individualprior to, during, or after the surgery. The inhibitor can beadministered parenterally, intravenous or injected into the tumor orsurrounding area after tumor removal.

Typically, one or more of the inhibitors of an SCD1 polypeptide providedherein can be formulated into a pharmaceutical composition that can beadministered to a mammal (e.g., rat, dog, horse, cat, mouse, rabbit,pig, cow, monkey, or human). For example, A939572 or a pharmaceuticallyacceptable salt thereof can be in a pharmaceutically acceptable carrieror diluent. A “pharmaceutically acceptable carrier” refers to anypharmaceutically acceptable solvent, suspending agent, or otherpharmacologically inert vehicle. Pharmaceutically acceptable carrierscan be liquid or solid, and can be selected with the planned manner ofadministration in mind so as to provide for the desired bulk,consistency, and other pertinent transport and chemical properties.Typical pharmaceutically acceptable carriers include, withoutlimitation, water, saline solutions, dimethyl sulfoxide, binding agents(e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers(e.g., lactose and other sugars, gelatin, or calcium sulfate),lubricants (e.g., starch, polyethylene glycol, or sodium acetate),disintegrates (e.g., starch or sodium starch glycolate), and wettingagents (e.g., sodium lauryl sulfate).

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of a compoundprovided herein. These salts can be prepared in situ during the finalisolation and purification of a compound provided herein, or byseparately reacting the compound in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, laurylsulphonate salts, and amino acidsalts, and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19.)

In some embodiments, a compound provided herein may contain one or moreacidic functional groups and, thus, is capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic inorganic and organic base additionsalts of a compound provided herein. These salts can likewise beprepared in situ during the final isolation and purification of thecompound, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate, orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary, ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum salts,and the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, and the like (see, forexample, Berge et al., supra).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES General Materials and Methods

Cell Culture

ccRCC cell lines: RWV366T and KIJ265T (16) (both stage 1V ccRCC patienttissue derived), A498, Caki1, Caki2, and ACHN (ATCC) and K347N, K355N,K359N, K360N, K365N, and K366N normal renal tissue derived mortal cells(NRE) were cultured in DMEM medium (Cellgro) containing 5% FBS (Hyclone)and 1× penicillin-streptomycin (Invitrogen) at 37° C. in humidifiedconditions with 5% CO₂.

Proliferation, Treatment, and Rescue Assays

Cells were plated (0.5 or 1×10⁵/well) in 24-well plates forproliferation or treatment assays, in triplicate. Cells were trypsinized(0.25%) and counted using a Coulter Particle Counter at specified timeintervals. For SCD1 rescue assays, oleic acid-albumin was added to mediaat 5 μM. Drug stocks were prepared in DMSO. Monotherapeutic treatmentidentified drug dose-response. Combinatorial dosing ranged up to theIC50 for each inhibitor. Temsirolimus dosing was performed as describedin the text. Soft agar cultures were prepared by diluting 2× growthmedium 1:1 in 1.5% Seaplaque®GTG® agarose, with 500 cells/plate in 60 mmculture dishes. Colonies were stained with Giemsa (LabChem. Inc.) andcounted after 3 wks. Cell images were obtained with an OlympusIX71microscope at 20× magnification.

Lentivirus

MISSION shRNA pLKO.1 constructs were used to make self-inactivatingshRNA lentiviruses for human SCD1 (clones:NM_(—)005063.3-1200s1c1[shSCD1200], NM_(—)005063.3-780s1c1[shSCD780]),and a non-target (NT) random scrambled sequence control (SHC002).Transfection reagents Lipofectamine 2000 and ViraPower were used togenerate lentiviruses using HEK293FT viral progenitor cells. ccRCC andNRE cells were incubated with lentivirus plus 5 μg/mL polybrene for 24hrs prior to clonal selection with Puromycin.

Transfections and Luciferase Assays

For transient transfection, Caki1 and A498 cells were transfected usingLipofectamine 2000. Cells treated with DMSO vs. A939572 or infectedusing shSCD780 lentiviral constructs vs. NT control were harvested after48 hrs using Promega's Dual Luciferase assay kit per the manufacturer'sprotocol and luciferase activity was measured using a VeritasLuminometer; reported as relative luminescence.

RNA Isolation and Quantitative PCR

An RNAqueous Midi Kit was utilized to extract and purify RNA from celllines. Human tissue RNA was prepared using TRIzol® per manufacturer'sprotocol followed by purification using the RNAqueous Midi Kit. The O.D.260/280 ratio of the mRNA was at least 1.8 and the 18 s/28 s bands wereverified on a 1% agarose gel. cDNA was prepared from purified RNAsamples using High Capacity cDNA Reverse Transcriptase Kit permanufacturer's instruction. TaqMan® Fast Universal PCR Master Mix andTaqMan® FAM™ dye-labeled probes including POLR2A (Hs00172187_m1)(normalization control), SCD1 (Hs01682761_m1), HSPA5 (Hs99999174_m1),CEBPβ (CEBPB Hs00270923_s1), GADD45A (Hs00169255_m1), DDIT3(Hs01090850_m1), and HERPUD1 (Hs01124269_m1) were combined with preparedcDNA samples to analyze relative mRNA expression via qPCR. Fold changevalues were compared between normal and tumor, non-target scrambledlentiviral and target lentiviral infected, and DMSO vs. A939572 treatedsamples using the ΔΔCt method (Schmittgen T D, Livak K J. Analyzingreal-time PCR data by the comparative C(T) method. Nat. Protoc. 2008;3:1101-8).

Gene Array Expression Analysis

Gene array expression analysis was performed using Affymetrix HumanGenome U133 Plus 2.0 Array chip. The details of the data processing andmethodology were previously described in (Tun H W, Marlow L A, vonRoemeling C A, Cooper S J, Kreinest P, Wu K, et al. Pathway signatureand cellular differentiation in clear cell renal cell carcinoma. PLoSOne. 2010; 5:e10696). Gene expression data was deposited at GeneExpression Omnibus (Accession#GSE41485). Pathway analysis was performedusing IPA (Ingenuity® Systems).

Western Blot Analysis

Protein extracts, electrophoresis, and membrane transfers were preparedas previously described (Copland J A, Marlow L A, Kurakata S, FujiwaraK, Wong A K, Kreinest P A, et al. Novel highaffinity PPARgamma agonistalone and in combination with paclitaxel inhibits human anaplasticthyroid carcinoma tumor growth via p21WAF1/CIP1. Oncogene. 2006;25:2304-17). Primary antibodies included SCD1, PARP, DDIT3, BiP, sXBP1,and β-actin. A Supersignal chemiluminescent kit was used to performdetection.

IHC and ICC Analysis

Formalin fixed, paraffin-embedded tissue microarray (TMA) of patientccRCC tumor and matched normal tissues and TMA of combinatorial in vivomouse tumor tissue. The TMAs were mounted on slides fromparaffin-embedded blocks according to IHC procedure and samples wereblocked with Diluent that contained Background Reducing Components(Dakocytomation) for 30 min and then probed for SCD1, Ki67, Caspase-3,CD31, phospho-mTOR, DDIT3, and XBP1. ICC preparation and staining wasperformed as previously described (Cooper S J, Von Roemeling C A, Kang KH, Marlow L A, Grebe S K, Menefee M E, et al. Reexpression of tumorsuppressor, sFRP1, leads to antitumor synergy of combined HDAC andmethyltransferase inhibitors in Chemoresistant cancers. Mol Cancer Ther.2012). Stain scoring was done using algorithms generated with Imagescopesoftware created by a histologist. H-scores were calculated based uponsignal intensity (0-3+) using the formula: [(1+%×1)+(2+%×2)+(3+%×3)],intensity (I)-scores were calculated by dividing signal intensity byarea, and nuclear (N)-scores were calculated by dividing % positivenuclei by total nuclei examined per area. Cases where insufficient tumortissue presented were excluded from the study. 20× images were obtainedusing Scanscope XT and Imagescope software. RWV366T cell line validationwas carried out as previously described (Cooper S J, Von Roemeling C A,Kang K H, Marlow L A, Grebe S K, Menefee M E, et al. Reexpression oftumor suppressor, sFRP1, leads to antitumor synergy of combined HDAC andmethyltransferase inhibitors in chemoresistant cancers. Mol Cancer Ther.2012).

In Vivo Analysis

A498 cells were subcutaneously implanted in athymic nu/nu mice at 1×10⁶cells/mouse in 50% Matrigel. Tumors reached ˜50 mm³ prior to treatment,which was carried out for 4 wks. A939572 was administered via oralfeeding using strawberry flavored Kool-Aid® in sterilized H₂O (0.2 g/mL)vehicle at 30 mg/kg in a 50 μl dose twice daily/mouse. Temsirolimus wassolubilized in 30% ethanol/saline and administered via intraperitonealinjection at 10 mg/kg in a 50 μl dose once every 72 hrs/mouse. Tumorvolumes were calculated using the formula 0.5236(L*W*H) and body weightwere measured every 3 days.

DNA isolation and STR Analysis

Genomic DNA was extracted from both RWV366T patient primary tissue andmatching cell line using Purelink™ Genomic DNA mini kit. Sixteen STRmarkers were PCR amplified using fluorescently labeled primers from ABI,and were analyzed using ABI 3130. Peak sizes were calculated versus aco-injected size standard using Gene Marker.

Statistical Analysis

Data values are presented as either percentage or fold change±s.d.unless otherwise specified. Fold change values 1.5< are consideredstatistically significant. Treatment group comparisons were analyzedusing two-tailed paired Student's t-test with p<0.05 being consideredstatistically significant. Statistically significant results areindicated by asterisk (*). Drug synergy statistics are indicated viacombination index (CI) determined using CalcuSyn® as described in thetext.

Example 1 SCD1 Polypeptide is Upregulated in ccRCC and is Involved inTumor Cell Survival

Normal kidney tissue and matched ccRCC tissue samples were obtained, andthe levels of SCD1 mRNA and SCD1 polypeptide expression were determined.SCD1 mRNA levels and SCD1 polypeptide levels were elevated in matchedccRCC samples when compared to normal samples (FIGS. 1A and 1B).

The levels of SCD1 mRNA and SCD1 polypeptide expression also weredetermined in established ccRCC cell lines and normal renal epithelialcells. SCD1 mRNA levels and SCD1 polypeptide levels were elevated inestablished ccRCC cell lines when compared to normal renal epithelialcells (FIGS. 1C and 1D). RWV366T is a newly established patient derivedccRCC cell line, whose patient and renal origins were validated by STRanalysis and IHC for renal markers (data not shown).

To determine the involvement of SCD1 expression in renal cell carcinomaproliferation, two separate lentiviral constructs were designed toexpress shRNA molecules having the ability to reduce SCD1 expression.The first lentiviral construct was designed to express an shRNAdesignated SCD780. The sequence of SCD780 was as follows:5′-CTACGGCTCTTTCTGATCATT-3′ (SEQ ID NO:1). The second lentiviralconstruct was designed to express an shRNA designated SCD1200. Thesequence of SCD1200 was as follows: 5′-CGTCCTTATGACAAGAACATT-3′ (SEQ IDNO:2). A non-target lentiviral construct was designed as a control.

Treatment of established ccRCC cell lines (Caki1 and A498) withlentiviral constructs designed to express SCD780 or SCD1200 resulted inreduced SCD1 mRNA expression levels (FIG. 2A) and reduced SCD1polypeptide expression levels (FIG. 2D). The reduction in SCD1expression in ccRCC cells revealed the induction of apoptosis asdemonstrated by poly ADP ribose polymerase (PARP) cleavage (FIG. 2D).Treatment of normal renal epithelial cells (K359N and K360N) withlentiviral constructs designed to express SCD780 or SCD1200 resulted inreduced SCD1 mRNA expression levels (FIG. 2A).

A proliferation assay was performed to determine if reduced SCD1expression preferentially reduced the ability of established ccRCC celllines to proliferate as compared to normal kidney cells. Treatment ofestablished ccRCC cell lines (Caki1 and A498) with lentiviral constructsdesigned to express SCD780 or SCD1200 resulted in reduced proliferationas compared to the levels of proliferation observed with normal kidneycells (K359N and K360N) treated with the lentiviral constructs (FIGS. 2Band 2C).

These results demonstrate that inhibitors of SCD1 can be used to reducethe number of ccRCC cells present within a mammal, while having littleor no effect on normal kidney cells. These results also demonstrate thatloss of SCD1 in ccRCC cells can lead to apoptotic programmed cell death.

Example 2 Oleic Acid Reverses Effects of Decreased SCD1 Expression inTumor Cells

As oleic acid (OA) is the principle product of SCD1 mediated SFAdehydrogenation, a cell culture stable form of OA conjugated to albuminfrom bovine serum (OA-BSA) was utilized to perform rescueexperimentation in order to confirm that decreased tumor cell growth andinduction of cell death was due to lentiviral mediated suppression ofSCD1.

Media alone and BSA supplemented media served as control groups.Proliferation assay of NT control versus shSCD780 infected Caki1 andA498 cells cultured in media with or without OABSA were counted afterfive days. Both Caki1 and A498 shSCD780 cells exhibited significantdecreases in growth when compared to controls; however the addition ofOA-BSA rescued the proliferative capacity of these cells to near controlrates (FIG. 3A). Notably, addition of OABSA to Caki1 NT cells marginallyenhanced proliferation (FIG. 3A). SCD1 knockdown by lentiviral infectionwas confirmed at the protein level (FIG. 3B). In addition to growthrescue, supplementation with OA-BSA also decreased shSCD780 inducedapoptosis as demonstrated by reduction in PARP cleavage shown by westernblot (FIG. 3B). Representative phase contrast images of ccRCC cells foreach group are shown in FIG. 3C.

Example 3 Small Molecule Inhibition of SCD1 Induces ccRCC Cell Death

A939572 was dosed out in four ccRCC cell lines—Caki1, A498, Caki2, andACHN, and demonstrated a significant dose-dependent decrease inproliferation at day 5 (IC50s of 65 nM, 50 nM, 65 nM, and 6 nM,respectively) (FIG. 4A). Molecular target specificity was confirmed byaddition of OA-BSA to the growth inhibitory assay, with IC50 dosesapplied to all four cell lines versus DMSO+BSA control. Addition ofOA-BSA prevented A939572 mediated growth inhibition which was comparableto control groups in all four cell lines (FIG. 4B). In congruity withprevious experimentation examining SCD1 lentiviral knockdown models,A939572 induced apoptosis confirmed by PARP cleavage via western blotanalysis in all four cell lines (FIG. 4C). Addition of OA-BSA blockedapoptosis noted by lack of PARP cleavage (FIG. 4C). Representative phasecontrast cell images (FIG. 4D) demonstrate marked reduction inconfluence of A939572 treated ccRCC cells (day 5), which reflectsdecreased proliferation and induction of cell death as a result oftreatment. OA-BSA supplemented cells display no visible alterations inphenotype. Thus, we have identified a specific small molecule SCD1inhibitor that induces apoptotic cell death that can be rescued by oleicacid.

Example 4 Treatment of ccRCC Cells with A939572 Induces EndoplasmicReticulum Stress

In order to determine the mechanism of decreased proliferation andinduction of cell death associated with loss of SCD1 activity in ccRCCcells, gene array analysis was performed with Caki1, A498, Caki2, andACHN ccRCC cells treated for 24 hours with a 75 nM dose of A939572compared to DMSO control. Gene expression data was analyzed using theIngenuity® Systems (IPA) program and revealed increased expression of ERstress response genes associated with UPR.

Western blot of Caki1 and A498 cells for protein expression of key ERstress markers including BiP (heat shock 70 kDa protein, GRP78), CHOP(DNA damage inducible transcript 3, DDIT3), and spliced-XBP1 (x-boxbinding protein 1, s-XBP1) revealed amplified expression in both drugtreated (75 nM) and shSCD780 lentiviral knockdown cells after 48 hours(FIG. 5A), confirming induction of ER stress upon loss of SCD1 activityor expression as implicated by the gene array analysis.

In order to validate the specificity of ER stress induction mediated byboth A939572 and shSCD780, rescue assays were performed using OA-BSA inCaki1 and A498 cells qPCR analysis of five ER stress genes identified inthe gene array including BiP, CHOP, HERPUD1 (homocysteine-inducible,ER-stress inducible, ubiquitin-like-1), GADD45a (DNA damage inducibletranscript 1, DDIT1), and CEBPβ (CCAAT/enhancer binding protein beta)were examined. In A939572 (SCDi) treated Caki1 and A498 cells, all fiveER stress related genes were expressed at significantly increased levelscompared to DMSO+BSA control, and this elevated expression could beblocked with the addition of OA-BSA (FIG. 5B). In shSCD780 lentiviralinfected Caki1 and A498 cells, all of the ER stress genes weresignificantly induced in the Caki1 shSCD780 sample and 4 of the 5 weresignificantly induced in the A498 shSCD780 sample. Similar to the drugtreated cells, OA-BSA successfully blocked shSCD780 induced expressionof the ER stress genes (FIG. 5B).

Activating transcription factor 6 (ATF6) is a key bZIP transcriptionfactor that mediates part of the UPR stress response. Upon stressinduction ATF6 is proteolytically cleaved into the activatedtranscription factor allowing it to transcribe several downstreammediators in the ER stress response pathway including XBP1, BiP, HSP90B1(heat shock protein 90 kDa beta), and CHOP (23). Caki1 and A498 cellstransfected with an ATF6 luciferase reporter (p5xATF6-GL3) were treatedwith a 75 nM dose of A939572 or were infected with shSCD780. Resultingluminescence was measured after 48 hours Inhibiting SCD1 genetically orpharmacologically resulted in significant enhancement of luciferaseactivity as compared to DMSO and NT controls with Caki1 A939572+BSA,Caki1 shSCD780+BSA, A498A939572+BSA, and A498shSCD780+BSA cellsexpressing fold change inductions of 1.6, 1.7, 3.8, and 2.0 respectively(FIG. 5C). The addition of OA-BSA significantly reduced reporteractivation in response to A939572 and shSCD780, thereby confirmingspecificity of ATF6 stimulation by loss of SCD1 activity in ccRCC cells.Collectively, these data are indicative that SCD1 inhibition activatesthe UPR stress response. Tumor cells may therefore be prone to elevatedlevels of ER stress requiring the induction of protective factors suchas SCD1 in order to preserve cell viability. Targeting ER protectiveconstituents presents another potential route for therapeuticintervention not only in ccRCC, but likely in other cancers as well.

Example 5 Combination of A939572 with Temsirolimus SynergisticallyEnhances Tumor Cell Death

In order to target ccRCC using a multifaceted approach, synergy wasexamined through application of combinatorial treatment utilizingA939572 in congruence with a current FDA approved regimen for ccRCCtreatment. These included the TKIs pazopanib and sunitinib, as well asthe mTOR inhibitor temsirolimus.

After identifying appropriate cell proliferative dose responses forpazopanib and sunitinib in four ccRCC cell lines including A498, Caki1,Caki2, and ACHN, both TKIs were dosed in combination with A939572 up toapproximately the IC50 dose for each drug in the Caki1 and the A498 celllines. No synergy was noted in either Caki1 or A498 cell proliferativeresponses with combinatorial treatment. Temsirolimus (Tem) when dosedout in the four ccRCC cell lines yielded a limited reduction in cellproliferation, and no dose response could be determined. Combinatorialtreatments were therefore done using a fixed dose of Tem (0.1 nM, 1 nM,and 10 nM) combined with a dose range of A939572 up to the IC50 inCaki1, A498, Caki2 and ACHN cells. Both drugs in combination yieldedvery strong synergy in all four cell lines as indicated by thecombination index (CI) determined using CalcuSyn® based on theChou-Talalay Method where CI values >1 represent an antagonistic effectand values <1 represent synergy, with lower values signifying enhancedsynergy. Colony formation assay of A498 cells grown in soft agar treatedwith mono and combination doses of 5 nM A939572 and 5 nM Tem reflectedsynergistic effects observed in combination growth assays performed in2-D culture and provided the rationale for in vivo analysis ofcombinatorial therapy.

Athymic nude (nu/nu) mice bearing A498 ccRCC xenografts were treatedwith A939572 and Tem individually or in combination over the course offour weeks, and tumor volume (mm₃) was recorded (FIG. 6A). A939572 andTem monotherapy generated similar growth responses with approximately20-30% reductions in tumor volume (vs. placebo control) being observedupon study completion, with values reaching statistical significanceonly within the last week of treatment. The combination group yieldedover a 60% decrease in tumor volume (vs. placebo control) by studycompletion with significant reductions recorded after approximately 1week of treatment. All of the animals maintained a healthy weightthroughout the course of the treatment (FIG. 6A), however those in boththe A939572 and the Combo group exhibited increased blinking, and slightmucosal discharge from the eyes after the first week of treatment.

IHC analysis of tumors resected from each treatment group was analyzedfor proliferation, angiogenesis, and cell death (FIG. 6B). All treatmentgroups (A939572, Tem, and Combo) when compared to the placebo controlexhibited decreased proliferation as marked by reduction in percentpositivity of nuclear Ki67 staining, with the combinatorial groupdemonstrating the most significant decline. Angiogenesis as examined byintensity of microvessel density demonstrated a slight decrease in boththe Tem and the Combo groups; however the cumulative scores were notconsidered significant. Cell death as examined by cleaved caspase-3(CC3) demonstrated significant increases in the Combo group whencompared to all groups. A moderate increase in cell death was also seenin the A939572 and Tem groups compared to the placebo. PhosphorylatedmTOR was inspected as a marker for temsirolimus activity, and decreasedexpression was confirmed in both the Tem and the Combo groups ascompared to the Placebo and A939572 groups. ER stress was examined viawestern blot of total protein extractions prepared from randomlyselected tumor tissue samples representing each treatment group, andresulting quantitative expression was normalized to respective βactincontrols. Increased expression of CHOP was confirmed in all samplestreated with A939572 (A939572 and Combo) (FIG. 6C) confirming thatinhibition of SCD1 in ccRCC contributes to ER stress in vivo. A proposedmechanism is summarized in FIG. 6D. Interestingly, samples in the Temgroup also exhibited induction of CHOP, although to a lesser extent whencompared to A939572 and Combo groups. Temsirolimus has been previouslyreported to decrease SCD1 expression in breast cancer cells Inhibitionof mTOR in ccRCC could indirectly mediate ER stress through decrease ofSCD1, thereby explaining our observations. No significant increase inCHOP expression was seen in any placebo samples, confirming specificityof ER stress induction as a result of drug treatment.

Example 6 Inhibition of SCD1 Polypeptide in Various Cancer Cell Lines

A number of cancer cell lines were tested to determine whether SCD1protein expression correlates with growth inhibition of an SCD1inhibitor in human cancer cell lines.

Pancreatic Cancer

Cells (20,000/ml) were plated in 12 well cell culture plates, allowed toattach and treated with the indicated dose of SCD1 inhibitor (A939753)or standard of care (gemcitabine). Cell number was counted using aCoulter Counter. As show in FIG. 7, the data are expressed as percent ofDMSO control. Each value represents triplicates. Western analysis forSCD1 protein expression was performed on each cell line with beta-actinas the loading control. The data indicated that MiaPaca cells expressSCD1 and were growth inhibited in a dose dependent fashion while Panccells expressed very low levels of SCD1 and were growth inhibited atonly high levels of SCD1 inhibitor, A939572.

The following cell lines were studied using a similar method as thatdescribed above.

Liver Cancer

It was found that SNU449 liver cancer cells express SCD1 protein and aregrowth inhibited in a dose dependent fashion by the SCD1 inhibitor,A939572 (see FIG. 8). An estimated IC₅₀ concentration occurred around100 nM. Sorafenib is FDA approved for liver cancer treatment and iseffective between 1-10 micromolar concentrations.

Melanoma

A375 melanoma cells express SCD1 protein and were growth inhibited in adose dependent fashion by the SCD1 inhibitor, A939572 (see FIG. 9). Anestimated IC50 concentration occurred around 50 nM. Mela 11 melanomacells do not express SCD1 and were not growth inhibited. Standard ofcare, Temodar, dose responsively inhibits growth in A375 cells but notMela 11.

Colon Cancer

Caco2 and HT29 colon cancer cells express SCD1 protein and are growthinhibited in a dose dependent fashion by the SCD1 inhibitor, A939572(see FIG. 10).

Bladder Cancer

T24 and HT1376 bladder cancer cells express SCD1 protein and were growthinhibited in a dose dependent fashion by the SCD1 inhibitor, A939572(see FIG. 11). The standard of care for bladder cancer, Cisplatin, hasminimal growth inhibitory effects on these two cell lines.

BCJ4T bladder cancer cells do not express SCD1 protein and were notgrowth inhibited by the SCD1 inhibitor, A939572 (see FIG. 12). Thestandard of care for bladder cancer, Cisplatin, has minimal growthinhibitory effects on these this cell line.

Anaplastic Thyroid Cancer

KTC3 thyroid cancer cells express SCD1 protein and were growth inhibitedin a dose dependent fashion by the SCD1 inhibitor, A939572 (see FIG.13). Taxol is growth inhibitory in KTC3 cells but has minimal growthinhibitory effects on FF1 cells.

Lung Cancer

A549 nonsmall cell lung cancer cells express SCD1 protein and weregrowth inhibited in a dose dependent fashion by the SCD1 inhibitor,A939572 while Calu-1 lung cancer cells do not express SCD1 and are notgrowth inhibited by A939572 (see FIG. 14). Taxol is growth inhibitory inA549 but was not tested in Calu-1 cells.

Ovarian Cancer

OVCA420 and HOV TAX2 ovarian cancer cells express SCD1 protein and weregrowth inhibited in a dose dependent fashion by the SCD1 inhibitor,A939572 while Calu-1 lung cancer cells do not express SCD1 and were notgrowth inhibited by A939572 (see FIG. 15). Taxol is growth inhibitory inHOV Tax2 cells but was not tested in OVCA420 cells.

Breast Cancer

MCF-7 (ER+/PR+), MDA-231 (triple negative) and T47D (PR+) breast cancercells express SCD1 protein and were growth inhibited in a dose dependentfashion by the SCD1 inhibitor, A939572 (see FIG. 16).

Prostate Cancer

DU-145 and LNCAP prostate cancer cells express SCD1 protein and weregrowth inhibited in a dose dependent fashion by the SCD1 inhibitor,A939572 (see FIG. 17).

Quantitation (Relative) of SCD1 Protein Expression in Different CancerCell Lines

As shown in FIG. 18, Western analysis was performed on SCD1 and betaactin. Quantitation was performed by first normalizing to eachrespective beta-actin followed by normalization to A498 control. SNU449cells appeared to have the highest SCD1 protein expression while BCJ4,Mela11 and PANC had the lowest protein levels. Protein levels appear tocorrelate with growth inhibition of the SCD1 inhibitor.

As shown in FIG. 19, Western analysis was also performed on SCD1 andbeta actin. Quantitation was performed by first normalizing to eachrespective beta-actin followed by normalization to A498 control. LN Capcells appeared to have the highest SCD1 protein expression while Calu1,FF1 and KTC3 cells had the lowest protein levels. Protein levels appearto correlate with growth inhibition of the SCD1 inhibitor.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for reducing the number of renal cellcarcinoma cells within a mammal, wherein said method comprisesadministering, to said mammal, an inhibitor of an SCD1 polypeptide andan inhibitor of an mTor polypeptide under conditions wherein the numberof viable renal cell carcinoma cells present within said mammal isreduced.
 2. The method of claim 1, wherein said inhibitor of an mTorpolypeptide is sirolimus (RAPAMUNE®), temsirolimus (CCI-779), everolimus(RAD001), or ridaforolimus (AP-23573).
 3. The method of claim 1, whereinsaid mammal is a human.
 4. The method of claim 1, wherein saidadministration is an intratumoral, oral, intraperitoneal, intramuscular,or intravenous administration.
 5. The method of claim 1, wherein saidinhibitor of an SCD1 polypeptide is A939572, MK-8245, CVT-11127, MF-152,or HYR-061.
 6. A method for reducing the number of renal cell carcinomacells within a mammal, wherein said method comprises administering, tosaid mammal, an inhibitor of an SCD1 polypeptide under conditionswherein the number of viable renal cell carcinoma cells present withinsaid mammal is reduced.
 7. The method of claim 6, wherein said mammal isa human.
 8. The method of claim 6, wherein said administration is anintratumoral, oral, intraperitoneal, intramuscular, or intravenousadministration.
 9. The method of claim 6, wherein said inhibitor isA939572, MK-8245, CVT-11127, MF-152, or HYR-061.
 10. The method of claim6, wherein said mammal is further administered an inhibitor of a mTorpolypeptide.
 11. The method of claim 10, wherein said inhibitor issirolimus (RAPAMUNE®), temsirolimus (CCI-779), everolimus (RAD001), orridaforolimus (AP-23573).
 12. A method for reducing the number of renalcell carcinoma cells within a mammal, wherein said method comprisesadministering, to said mammal, a composition under conditions whereinthe number of viable renal cell carcinoma cells present within saidmammal is reduced, wherein said composition comprises the ability toreduce SCD1 mRNA expression or SCD1 polypeptide expression.
 13. Themethod of claim 12, wherein said mammal is a human.
 14. The method ofclaim 12, wherein said administration is an intratumoral, oral,intraperitoneal, intramuscular, or intravenous administration.
 15. Themethod of claim 12, wherein said composition comprises a nucleic acidconstruct having the ability to express an shRNA directed against SCD1nucleic acid.